Hemolytic potential of miltefosine is dependent on cell concentration: Implications for in vitro cell cytotoxicity assays and pharmacokinetic data

Abstract

Miltefosine possesses antiparasitic, antibacterial, antifungal and antitumor activities; however, its mechanism of action is not well established. In the current work, the miltefosine concentrations required to achieve 50% hemolysis in PBS were shown to vary from 600 μM using 5×10(9) cells/mL to ~2.9 μM for ~5×10(6) cells/mL. This cell concentration-dependent hemolytic potential was described using an equation that included the membrane-water partition coefficient (K) and miltefosine concentrations in the cell membrane (cm) and aqueous medium (cw) as variables. The best-fit values for the 50% hemolysis data were log K=4.68, cm=110.8 mM, and cw=2.3 μM. Hemolysis measurements in whole blood were used to determine the erythrocyte membrane-plasma partition coefficient of miltefosine (Log KM/P=1.77). Additionally, miltefosine concentration in whole blood was found to be ~86% of that in plasma. Previously reported clinical pharmacokinetics data indicate that the plasma concentration of miltefosine peaks at ~90 μg/mL when treating visceral leishmaniasis. Using this concentration, which corresponds to ~77 μg/mL miltefosine in whole blood, we found only 2.8% hemolysis. Significant hemolysis (5.4%) was observed only after doubling the concentration to 180 μg/mL. Recently reported data indicate that miltefosine inhibitory concentrations in Leishmania are also dependent on cell concentration. The biophysical parameters assessed in the current study indicated that this type of response is associated with the accumulation of the drug in the cell membrane, which becomes damaged when critical drug concentrations are reached.

Dual Gene Delivery Reagents From Antiproliferative Alkylphospholipids for Combined Antitumor Therapy

“…The concentration provoking 50% hemolysis (HC 50 , calculated by using the best-fit regression curve method) was 43 and 46 μM for miltefosine and perifosine, respectively ( Figure 6 ). The HC 50 value found for miltefosine was consistent with those reported in the literature (41 μM Petit et al, 2019 , and 34–51 μM Alonso and Alonso, 2016 , depending on the hematocrit). Though other authors have reported ca .…”

Incorporation of the antitumor drug miltefosine into polymeric micelles

“…In addition, by interpolating the linear regression model of Figure 7A, incorporation of up to 300 µg/mL HePC in 4.5% F127 polymeric micelles can be considered safe in terms of hemolytic effect. Since HePC hemolytic activity is dependent on RBC concentration (Alonso and Alonso, 2016) and assuming a 40% hematocrit, it is possible to inject up to 4 mg/mL of HePC formulated in 4.5% F127 polymeric micelles. If we consider that plasmatic proteins provide extra protection to HePC action into erythrocytes, the amount of drug to be incorporated for bolus administration in whole blood could be even higher.…”

“…Among these, we highlight miltefosine (hexadecylphosphocholine, HePC, Figure 1A), the main representative of the class of anti-tumor synthetic alkylphospholipids (ALPs) clinically approved by the FDA for topical treatment of skin metastases of breast cancer, as well as cutaneous lymphoma . HePC is also widely used as an effective peroral treatment for visceral leishmaniasis (Alonso and Alonso, 2016). One of the most studied alternatives to reduce drugs hemolytic effect is nanoencapsulation and an interesting alternative refers to polymeric micelles, which are colloidal nanostructures (~10-100 nm) of amphiphilic block copolymers, which spontaneously self-aggregate in aqueous environments (Rangel-Yagui et al, 2005).…”

“…Akt-mTOR inactivation), lipid-dependent signal transduction (e.g. phospholipase C, phospholipase D, protein kinase C), as well as stress pathways leading to apoptosis (Van Blitterswijk and Verheij, 2008;Sá and Rangel-Yagui 2015;Alonso and Alonso, 2016).…”

“…HePC displays potent antitumor activity in vitro and in vivo; it is clinically approved by the FDA for topical treatment of skin metastases of breast cancer, as well as cutaneous lymphoma. HePC is widely used as an effective peroral treatment for visceral leishmaniasis and it has been reported to possess similar modes of action in Leishmania and human cancer cells (Alonso and Alonso, 2016). It also exhibits antibacterial, anti-trypanosomal and broad-spectrum antifungal activities .…”